Connecting TDP-43 Pathobiology to the Molecular Profiles of TDP-43 Driven Common Dementias In previous collaborative work[1], Dr. Phatnani and Dr. Gale-Hammell used a large ALS patient sequencing consortium to uncover the predominant molecular pathways that are altered in the frontal and motor cortex of patients with this TDP-43 associated disease. In this work, ALS cortex samples could be differentiated into 3 distinct groups based upon whether their transcriptional profiles showed hallmarks of: oxidative stress, retrotransposon de-silencing, or neuroinflammation. Histological staining of tissues from these same patients showed tight links between TDP-43 proteinopathy and the latter two groups of patients, though TDP-43 protein aggregates were most predominant in the retrotransposon expressing subset. This grant will build on our prior work to address whether similar TDP-43 dependent pathomechanisms are involved in common dementias associated with TDP-43 dysfunction, specifically frontotemporal dementia with TDP-43 proteinopathy (FTD-TDP) and Alzheimer?s Disease (AD). Our hypothesis is that retrotransposons contribute in part to the toxicity associated with TDP-43 dysfunction in FTD-TDP and AD, which we will conclusively test in this proposal. We have amassed a large collection of tissue samples from over 200 patients with either FTD-TDP or AD, and with extensive clincopathological characterization. The profiles from these samples will be used to guide deep mechanistic studies into the pathomechanisms of TDP-43 in FTD and AD. Aim 1: How does the cell- and tissue-specific context of TDP-43 proteinopathy affect its impact in FTD and AD? We will use multiplexed immunostaining data of postmortem cortical tissue from 200 FTLD-TDP and AD patients selected to show signatures of TDP-43 pathology, and in age-matched sudden death controls. We will obtain high spatial resolution IHC images of multiple independent factors, including pathological markers (pTDP43 and pTau), markers of cell type, and components of pathways identified to be coincident with TDP-43 dysfunction in our previous work such as inflammatory markers (IBA1, TREM2) and retrotransposon proteins (HERVK-env and L1HS-orf1). At adjacent slices from the same tissue, we will use Spatial Transcriptomics (ST) to define a high-resolution transcriptomic atlas of cell-type specific and context-specific impacts of TDP-43 proteinopathy. These combined molecular profiles will allow us to directly assess the contribution of TDP-43 dysfunction to cell-type specific and proteinopathy-proximal effects. Aim 2: Do active retrotransposons simply report on TDP-43 dysfunction or contribute to cellular toxicity in FTD and AD? We will obtain long read PacBio sequencing of individual expressed retrotransposons in FTD-TDP and AD patient samples to robustly determine the specific expressed genomic retrotransposon loci in both dementias, and their relative ability to produce functional proteins. We will then use CRISPRa to activate individual retrotransposons identified above in iPS differentiated cortical neurons to test for relative cellular toxicity. In the converse experiment, we will use CRISPRi to inhibit the activity of individual retrotransposons identified above in iPS neurons with TDP-43 dysfunction to test for the relative ability to alleviate TDP-43 mediated neurotoxicity. Aim 3: Can TDP-43 dependent retrotransposons contribute to activation of astrocytes and microglia? In the NYGC ALS Consortium patients, we previously saw activation of innate immune signaling pathways in samples with evidence of TDP-43 proteinopathy and activated microglia. Moreover, retrotransposons have previously been shown to activate innate immune pathways in other contexts. This aim will establish whether TDP-43 dependent retrotransposons are sufficient to induce activation of adjacent glial cells in cellular models of FTD-TDP and AD. We will express individual retrotransposons in iPS derived cortical neurons and test for the activation of adjacent astrocytes and microglia in a 3D organoid co-culture system. In the converse experiment, we will express TDP-43 dependent retrotransposons in glial cell types, to test whether retrotransposons more potently activate innate immune signaling pathways in astrocytes and microglia, which subsequently secrete neurotoxic factors.